Live yeast dietary supplementation acts upon intestinal morpho-functional aspects and growth in weanling piglets

Animal Feed Science and Technology 129 (2006) 224–236

Live yeast dietary supplementation acts upon intestinal morpho-functional aspects and growth in weanling piglets V. Bontempo ∗ , A. Di Giancamillo, G. Savoini, V. Dell’Orto, C. Domeneghini Department of Veterinary Sciences and Technologies for Food Safety, University of Milan, Via Celoria 10, 20133 Milan, Italy Received 10 September 2004; received in revised form 6 December 2005; accepted 29 December 2005

Abstract Three hundred and fifty-two piglets were at weaning assigned to two dietary experimental groups, Ctr (control) or Y (yeast-supplemented). The yeast supplement (2 g/kg of diet) provided 2×106 CFU/g of feed. Piglet growth was monitored from weaning to 4 weeks after weaning. On day 28 post-weaning, 20 female piglets (10 per group) were slaughtered and the distal ileum was sampled from each animal, and examined with microanatomical methods (histology, histochemistry, immunohistochemistry, and histometry). Villus and crypt measurements were taken, mucin profile assessed with histochemistry, and mucosal macrophages and proliferating epithelial cells determined after immunolocalization. Yeast supplementation to piglets was associated with a greater live weight (P<0.001) and a greater postweaning daily gain (P<0.001) in comparison with control animals. The intestinal adherent mucous layer was thicker in control than supplemented piglets (P<0.001). Proliferating epithelial cell counts were greater in supplemented than control piglets (P=0.045). Mucosal macrophages were more numerous in supplemented than control piglets (P=0.007). These findings indicate that live yeast dietary supplementation to piglets is able to improve nursery growth performance. Effects on intestinal mucosa suggest that supplementation is able to promote a “healthy” intestine, encouraging an early restora-

∗

Corresponding author. Tel.: +39 02 58357900; fax: +39 02 58357898. E-mail address: [email protected] (V. Bontempo).

0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2005.12.015

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tion of the intestinal mucosal thinning that often occurs at weaning, and possibly improving local resistance to infection. © 2006 Elsevier B.V. All rights reserved. Keywords: Live yeast; Piglet; Ileum; Mucine profile; Immunohistochemistry; Histometry

1. Introduction Piglets undergo a stress-related growth check at weaning, often associated with anorexia and under-nutrition, with predisposition to diarrhoea and intestinal infections. Several alternatives to antibiotics have been proposed for managing piglet gut health at this crucial period, including the administration of probiotics. Probiotics are preparations of non-pathogenic microorganisms, prepared for animal and human use, that may have beneficial effects on the digestive ecosystem and confer resistance to infection (Fuller, 1992). Live yeast preparations are probiotics rich in enzymes, vitamins, nutrients and co-factors. Yeasts are highly resistant to inactivation during their passage along the alimentary canal, and in humans may colonize the gut to help in restoring disturbed gastrointestinal microecology (Girola and Ventura, 1995; Bleichner et al., 1997). They have been reported to produce a variety of beneficial production responses in animals, including piglets (Jurgens et al., 1997; Maloney et al., 1998; Mathew et al., 1998; Van Heugten et al., 2003). However, other studies have reported the absence of benefits from live yeast supplementation (Kornegay et al., 1995). Saccharomyces cerevisiae ssp. boulardii is a non-pathogenic yeast that, when orally given to laboratory mammals, has been reported to stimulate gut-associated-lymphoid tissue (GALT) resulting in enhanced secretion of specific IgA (Buts et al., 1990), to inhibit binding of bacterial toxins to enterocyte receptors (Pothoulakis et al., 1993), and to enhance endoluminal production of polyamines thereby reducing intestinal permeability (Kollman et al., 2001; Costalos et al., 2003; Buts et al., 1994). The aim of the present study was to investigate the effects of dietary supplementation with live S. boulardii on morpho-functional aspects of piglet intestine, and on aspects of piglet performance during the first month after weaning. 2. Materials and methods A total of 352 piglets from weaning (25 day of age) until a month after weaning were used under farm conditions. Piglets were weaned by moving them to environmentally controlled pens. There were 16 pens with 20–25 piglets per pen; males and females were separated. Each pen was 0.9 m×2 m, had a slatted floor, and was equipped with water nipple and four-hole self-feeder. The piglets were allowed an ad libitum access to feed and water. 2.1. Diet Piglets were randomly allotted to control (Ctr) or yeast dietary supplementation (Y) with 8 replicates per treatment. The yeast supplement used was a concentrate of live S. cerevisiae

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Table 1 Composition of piglet diet, as-fed basis Ingredients

g/kg

Ingredients Maize Barley Cereals, steam-rolleda Milk powder, spray-dried Whey powder, spray-dried Full fat whey (50% fat) Fish meal, menhaden Soybean meal (48% CP) Maize oil Coconut oil Dextrose Limestone, ground Dicalcium phosphate Salt Vitamin–mineral premixb Copper sulfate Zinc oxide l-Lysine HCl dl-Methionine Tryptophan Yeastc

356 50 85 127 103 15 75 130 25 5 10 7 5 1 2.6 0.8 0.3 1.5 0.6 0.2 +/−

Calculated composition ME (MJ/kg) CP (g/kg) Lysine (g/kg) Ca (g/kg) P (g/kg)

13.8 210.2 14.1 9.1 7.5

a

Mixed cereals: 250 g maize, barley, oats and wheat/kg. Supplying a minimum per kilogram complete diet of: 12,500 IU Vitamin A; 1250 IU Vitamin D; 125 IU Vitamin E; 90 ␮g Vitamin B12 ; 9 mg riboflavin; 45 mg pantothenic acid; 35 mg niacin; 4.5 mg folic acid; 0.25 mg biotin; 130 mg Fe; 170 mg Zn; 15 mg Cu; 30 mg Mn; 0.60 mg I; and 0.28 mg Se. c Live yeast (Saccharomyces cerevisiae ssp. boulardii, Levucell SB, CNCM I-1079; Lallemand, France) was added to treated diets at 2 g/kg of feed at the expense of maize steam rolled or corn alone (calculated as 2×106 CFU/g of feed). b

ssp. boulardii (Levucell SB-CNCM I-1079, Lallemand, France). Live yeast content was over 2×106 CFU/g of feed. All piglets received a starter diet that was either control (Ctr, no added yeast) or contained 2 g/kg added live yeast (Y, 2×106 CFU/g of feed). Diets were fortified to meet or exceed NRC (1998) requirements for all nutrients (Table 1). Antibiotics as growth-promoting agents were absent. Total zinc and copper were present in the diet at 270 and 30 ppm, respectively. Live weight, feed intake, and feed efficiency were recorded for the 28-day post-weaning study. All animals were treated in accordance with European Community guidelines approved by the Italian Ministry of Health.

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2.2. Microscopic anatomy of piglet ileum At day 28 post-weaning, 10 female piglets per group (total number of animals = 20) were slaughtered. Females were chosen for the intestinal microscopic study because of the homogeneous weight within the experimental groups. The entire intestinal tracts were removed, and the distal ileum (2 cm prior to the opening into the caecum) was collected from each animal (total number of samples = 20) and promptly fixed in 4% paraformaldehyde in 0.01 M phosphate buffered saline (PBS) pH 7.4 for 24 h at 4 ◦ C. The specimens were then dehydrated in graded alcohols, cleared with xylene and embedded in paraffin. After dewaxing and re-hydration, serial microtome sections (4 ␮m thick) were stained with hematoxylin and eosin and examined to assess microanatomical structure and determine villus height (V) (10 villi measured per section), crypt depth (C) (10 crypts measured per section), and the villus height to crypt depth ratio (V:C ratio). The ileum mucin profile was determined by staining some sections (a) with the Alcian blue 8GX, pH 2.5-periodic acid Schiff combination (AB-PAS), which reveals neutral (PASreactive) and acid (AB-reactive) glycoconjugates, and (b) with the high iron diamine-Alcian blue 8GX, pH 2.5 combination (HID-AB), which demonstrates sulphated and sialylated glycoconjugates respectively. The thickness of the adherent mucous layer, determined as the distance from the outermost surface of it to the luminal surface of the epithelial lining, was measured at 10 randomly selected points in each AB-PAS-stained section (Matsuo et al., 1997). Other ileum sections were processed to visualize mucosal cells, which were in the S phase of the cell cycle (proliferating cells), by immunostaining with a monoclonal antiserum against proliferating cell nuclear antigen (PCNA) (PC10 clone, Sigma, Italy), and the subsequent revelation of the immunoreactive sites with the peroxidase-antiperoxidase (PAP) method. Briefly, following dewaxing and re-hydration, the sections were immersed in a freshly prepared solution of 3% H2 O2 in distilled water for 10 min to block endogenous peroxidase activity. For the antigen retrieval, slides with sections were heated in a microwave at 700 W twice (5 min each) in citrate buffer 0.01 M, adjusted to pH 6.0 with NaOH 2N (Foley et al., 1991; Greenwell et al., 1991; Cattoretti et al., 1993; Shi et al., 1995). After cooling at room temperature (15 min), the sections were rinsed in Tris-buffered saline (TBS) pH 7.5 and incubated with normal swine serum (Dako, Italy) at 1:5 dilution in TBS containing 1% bovine serum albumin (BSA) for 20 min. The PCNA-antiserum was applied (dilution 1:3000 in TBS plus 1% BSA) for 45 min at room temperature in a humid chamber. Sections were then incubated with rabbit anti-mouse IgG (Dako), followed by incubation with mouse PAP complex (Dako). Immunoreactivity was visualized using a freshly prepared solution of 3,3 -diaminobenzidine tetrahydrochloride (Sigma). Sections were briefly counterstained with Mayer’s hematoxylin, dehydrated and permanently mounted with Eukitt (Bio Optica, Italy). The proliferative index was determined by counting epithelial cells with PCNA-positive nuclei in 10 well-oriented villi/crypts for each ileum section (Burrin et al., 2000a). Other ileum sections were processed immunohistochemically to identify mucosal macrophages using an anti-human macrophage monoclonal antiserum (LN-5 clone, Sigma) diluted 1:400 in TBS. The steps were as for PCNA immunostaining, except that antigen retrieval was not necessary. The macrophage index was determined in GALT. For each sec-

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Table 2 Least mean square of piglets growth performance (mean ± S.E.) Ctr (n = 176) Piglets weight (kg) At weaning 30-day post-weaning Average daily gain (g) Daily feed intake (g) Feed:gain

7.9 19.4 432 691 1.6

± ± ± ± ±

0.11 0.15 0.01 0.13 0.17

Y (n = 176) 7.5 20.4 474 670 1.4

± ± ± ± ±

0.11 0.14 0.01 0.12 0.15

P 0.019 <0.001 <0.001 0.110 0.180

tion, the number of immunopositive mucosal cells was counted in 10 fields (representing a tissue area of about 0.015 mm2 ) (Sozmen et al., 1996). The specificity of immunostaining was verified for both antisera by incubating other ileum sections with normal mouse serum (Dako) instead of the primary antisera: this always gave negative results. As positive controls, alimentary canal samples from cow and dog were tested. In all cases the expected positive reactions were observed. Slides from all groups were stained together in a single batch. For all the observations and measurements, an Olympus BX51 microscope equipped with DP software for the computed image analysis (Olympus, Italy) was used. Observations and counting were performed by an observer blind to the control versus yeast-supplemented status of the sections. 2.3. Statistical analysis ANOVA (SAS Inst., 1999) was used to analyze differences in piglet weight and average daily gain, including dietary supplementation, and pen within supplementation as fixed effects, and weight at weaning as covariate. Following which, piglets’ diet and pen within diet were analyzed by orthogonal contrast. Histometric data (villus height, crypt depth, V:C ratio, mucous layer thickness) were analyzed by ANOVA using the mixed procedure of the SAS package (1999). The model included the treatment as fixed effect and piglet as random effect. The total number of mucosal cells with PCNA-positive nuclei were added as a linear regression in the model. The total number of mucosal cells with anti-macrophage-positivity was also added as a linear regression in the model. The data are presented as least squared mean ± S.E.M. Differences between least squared means were analyzed by orthogonal contrast and considered significant at P<0.05.

3. Results 3.1. Effects of yeast supplementation on performance The effects of yeast supplementation on piglet growth are shown in Table 2. Control piglets were heavier (P<0.05) than treated piglets at weaning, but the latter ones were significantly heavier at 30 days post-weaning (P<0.01). Piglets fed yeast had a significantly

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Table 3 Villus height (V), crypt depth (C), V:C ratio; mucin profile; mitotic cell counts; mucosal macrophage counts in piglets ileum (mean ± pooled S.E.) Ctr (n = 10) Villous height (V) (␮m) Crypts depth (C) (␮m) V:C ratio Adherent mucous gel thickness (␮m) Mitotic cells % of total Macrophage

195 130 1.5 2.9 42 4.0

± ± ± ± ± ±

3 2 0.01 0.09 2 0.06

Y (n = 10) 243 177 1.4 1.7 49 4.9

± ± ± ± ± ±

3 2 0.01 0.08 2 0.08

P 0.001 0.001 0.088 <0.001 0.045 0.007

greater average daily gain from weaning throughout 30 days post-weaning (P<0.01) than non-supplemented piglets. Piglets fed yeast also grew more efficiently (better feed:gain ratio) than those fed basal diet from weaning through 30 days post-weaning, but the difference was not significant (Table 2). 3.2. Microscopic anatomy of piglet ileum Features suggesting histopathological aspects in the ileum of either control or treated piglets were never observed. The intestinal mucosa was regularly organized in intestinal villi and crypts in both control and yeast-supplemented piglets. The presence of GALT was usual for this species in both its prominence and localization (Peyer’s patches). Histometric analysis showed that villus height (P<0.01) and crypt depth (P<0.01) were significantly greater in yeast-fed piglets than those not given yeast. Consequently, V:C ratio was lower (P=0.088) in yeast-fed piglets than those not given yeast (Table 3).

Fig. 1. Control piglet ileum. PCNA immunostaining, scale bar: 20 ␮m. Positive nuclei are present in enterocytes of intestinal crypts (arrows).

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Fig. 2. Yeast-supplemented piglet ileum. PCNA immunostaining, scale bar: 20 ␮m. Numerous strongly immunoreactive nuclei are present in enterocytes of intestinal crypts (arrows).

AB-PAS sequential staining showed that intestinal goblet cells contained both neutral and acid glycoconjugates. Acid glycoconjugates were predominant in supplemented (Y) piglets, particularly in crypts. HID-AB staining showed that, in all cases, goblet cells containing sulphated glycoconjugates predominated in villi, and goblet cells containing sialylated glycoconjugates occurred mainly at the bases of crypts. In AB-PAS-stained sections, the adherent mucous gel was significantly thicker in control piglets than supplemented animals (P<0.01).

Fig. 3. Control piglet ileum. Macrophage immunostaining, scale bar: 20 ␮m. Immunopositive cells are present in the tunica propria (arrows).

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Fig. 4. Yeast-supplemented piglet ileum. Macrophage immunostaining, scale bar: 20 ␮m. Immunopositive cells are present in diffuse lymphatic tissue (arrows).

PCNA-immunoreactive nuclei were numerous in enterocytes of ileum samples from all groups (Figs. 1 and 2). Epithelial cells with such immunoreactive nuclei (proliferating cells) were significantly (P<0.05) more numerous in supplemented than control piglets. Immunostaining for macrophages picked out numerous rounded immunoreactive cells in the GALT of all animals (Figs. 3 and 4). However, macrophages were significantly (P<0.01) more numerous in treated than non-treated piglets.

4. Discussion The aim of the experiment was to determine the effects of dietary supplementation with live yeast (S. cerevisiae ssp. boulardii) on piglet growth and gut structure. It was particularly concerned with weanling piglets since these animals undergo a stress-related growth check at this time, and any reduction in gut tissue mass, linked for example to a gastrointestinal disorder (Burrin et al., 2000b), can result in marked morbidity and significantly reduced growth performance (Pluske et al., 1997). During post-weaning, piglets fed yeast had significantly greater average daily gain than control piglets, and this may be reasonably attributed to the yeast supplementation. Similar improvements in piglet average daily gain as a result of yeast supplementation have been reported by some studies (Jurgens et al., 1997; Maloney et al., 1998; Mathew et al., 1998), while others have found that yeast supplementation to weanling piglets had no effect on average daily gain (Kornegay et al., 1995; Jurgens et al., 1997). These discrepant findings may be due to differences in sanitary conditions of animals, composition of diet, and quantity and type of live yeast added to diet. Van Heugten et al. (2003) founded that live yeast supplementation had positive effects on nursery piglet performance, but only when diets contained growth-promoting anti-microbial substances.

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Histometric analysis showed that villus height and crypt depth were greater and V:C ratio was smaller in treated piglets than controls. Villus height and crypt depth are indirect indications of the maturity and functional capacity of enterocytes, and more long the villi and crypts are, a greater number of enterocytes are there present (Hampson, 1986). Baum et al. (2002) also found that villus length was greater in the small intestine of piglets fed yeast than controls. In poultry fed yeast, Bradley et al. (1994) found that villus height did not differ from controls, while Zaouche et al. (2000) found differences in the intestinal mucosa in rats given S. boulardii orally. These contrasting results suggest species-specific differences in response to S. boulardii supplementation. Considering the multiple functions of the glycoconjugates that make up intestinal mucins, and their differences in health and disease, at least in humans (Rhodes, 1989), we have assessed the mucin content of goblet cells and thickness of the adherent mucous gel. Acid glycoconjugates seemed more abundant in the mucous cells of supplemented than control piglets. In both treated and control animals sulphated acid glycoconjugates were abundant in mucous cells of the villi, whereas sialylated glycoconjugates were abundant in mucus cells of crypts. Sulphated glycoconjugates confer a high viscosity to intestinal mucins, which in such a dense status are potentially able to trap pathogenic bacteria, and so the localization of sulphated glycoconjugates at the surface of the intestinal mucosa is potentially very useful in the view of ensuring a local defensive response. Acid glycoconjugates, taken as a whole, help the intestinal mucosa to counteract microorganisms and resist to bacterial enzymes (Deplancke and Gaskins, 2001; Montagne et al., 2004). Increased quantities of acid glycoconjugates in yeast-supplemented piglets may confer a greater resistance to bacterial infection in the gut in comparison with controls. The histological and histochemical approaches to the quantitative analysis of the intestinal adherent mucous gel in pathological as well as experimental conditions is now accepted as correct, and in some instances preferred to the biochemical one (Sakamoto et al., 2000). Mucous layer is both a barrier and an interface between the lumen and the intestinal epithelium; it is also a lubricant and stabilizer of the intestinal microclimate as well as a source of energy for the resident microflora (Deplancke and Gaskins, 2001). In healthy animals, the adherent mucous gel produced by goblet cells prevents gut pathogens from invading the mucosa (Neutra and Forstner, 1987). In turn, pathogenic microbes secrete glycosidases and peptidases to degrade the mucus and gain access to the epithelial cells. Goblet cells usually step up glycoconjugate production in response to the presence of potential pathogens (Hoskins et al., 1985; Carlstedt-Duke et al., 1986). Mucous secretion is stimulated in laboratory mammals by bacterial infection (Cohen et al., 1983; Mantle et al., 1989), bacterial toxins (Roomi et al., 1984), and parasite infestation (Miller et al., 1981). In the present study, the intestinal mucous layer was thicker in control piglets than in supplemented animals (P<0.01). This may indicate a greater presence of potential pathogens in the gut lumen of non-supplemented piglets, suggesting that yeast supplementation is able to reduce levels of potential pathogens in the gut. Among the possible mechanisms of this effect, the potential production of microbial growth inhibitors by the yeast is to be mentioned. Talarico et al. (1988) showed that the probiotic microorganism Lactobacillus reuteri synthesizes a microbial growth inhibitor. Tang et al. (1999) also suggested a relation

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between a thick mucous gel and an increased presence of potential pathogens in piglet intestine. The thicker mucus layer detected in control animals may also limit the diffusion of nutrients to the apical surface of epithelial cells, thereby reducing absorption. Thus, the thinner mucus layer in yeast-treated piglets may have contributed to the better feed efficiency found in them in comparison with control animals, as also suggested by Patience et al. (1997). Proliferating cell counts were higher in the mucosa of supplemented piglets than controls. Enterocytes are constantly replaced by division of epithelial cells in crypts. The rate of replacement usually matches the rate of loss. Epithelial cells near villus tips are the most mature and have the greatest digestive and absorptive capacities (Aitken, 1984). In rats fed a probiotic preparation of Lactobacillus casei and Clostridium butyricum, Ichikawa et al. (1999) reported findings related to proliferating cells similar to those presented here, whereas Baum et al. (2002) found no difference between control weaned piglets and those treated with S. boulardii or Bacillus cereus var. Toyoi in terms of number of cells whose nuclei were in S phase. The greater proliferation rate of intestinal epithelial cells in treated piglets may be due to the enhanced endoluminal release of polyamines linked to the presence of yeast in the lumen (Buts et al., 1994; Costalos et al., 2003). Polyamines are necessary for cell growth and differentiation, and the demand for them is presumably high in rapidly proliferating tissues with a high rate of cell turnover such as the intestinal mucosa of a young animal, instances in which the intracellular concentrations of these substances increase (Tabor and Tabor, 1984; Alarcon et al., 1987; Costalos et al., 2003). It is conceivable that yeast-supplemented piglets are potentially able to restore the mucosal thinning that occurs at weaning (Van Beers-Schreurs et al., 1998) with a better efficiency than control piglets, as also suggested by Isolauri et al. (2001). This putative effect is not, however, accompanied by negative consequences on the intestine, since histological features suggesting pathological changes were never observed. Similarly, Kollman et al. (2001) found no evidence of intestinal hyperplasia in rats after administration of S. boulardii. Mucosal macrophages were more numerous in supplemented than control piglets. This suggests that, in the absence of any evidence of pathological changes in the gut, components of the innate defensive system may be more active in treated animals. Since the early phases of viral infections are mainly counteracted by macrophages (Kosugi et al., 2002), it is conceivable that supplemented piglets are protected against viral infections at a stronger extent than controls. In conclusion, S. boulardii supplementation is widely used in humans and food animals. The results of the experiment have shown that dietary supplementation is able to modify morpho-functional aspects of the ileum mucosa in piglets without producing pathological or other detrimental changes, and the observed modifications may suggest a way of action of this probiotic. These modifications may enable the animals to rapidly overcome some of the negative consequences of weaning, particularly the largely described mucosal thinning and increased susceptibility to gastrointestinal disorders. In addition, dietary supplementation with live S. boulardii seems to be useful in promoting intestinal health, and improving piglet growth.

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Acknowledgements Authors wish to thank Dr. Eric Chevaux (Lallemand Sas, France) for cooperation and providing yeast. Authors are also grateful to Giuseppe Cerri (Azienda Agricola Cavagnone, Buronzo, Italy) for providing facilities for the experiment.

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